Inhibitors of NF-kappaB activation

ABSTRACT

The present invention relates to novel inhibitors of the Nuclear factor kappa B (NF-κB) activating pathway useful in the treatment of NF-κB related diseases and/or in the improvement of anti-tumor treatments. These inhibitors interfere early in the TRAF induced signaling pathway and are therefore more specific than IκB.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/702,953,filed Oct. 31, 2000, now U.S. Pat. No. 6,673,897, which is acontinuation of International Application No. PCT/BE99/00055, filed May5, 1999, designating the United States of America, published in Englishas WO 99/57133, the contents of both of which are incorporated by thisreference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing appendix and a computerreadable form, both of which are hereby incorporatied by reference.

TECHNICAL FIELD

This invention relates generally to biotechnology, and more particularlyto novel inhibitors of the Nuclear factor kappa B (NF-κB) activatingpathway useful in the treatment of NF-κB related diseases and/or in theimprovement of anti-tumor treatments. The invention also relates tonucleic acids coding for the novel inhibitors. The invention relatesfurther to the use of polypeptides, derived from these inhibitors in thetreatment of NF-κB related diseases and/or cancer. Furthermore, theinvention concerns pharmaceutical preparations, comprising the novelinhibitors or the polypeptides, derived from these inhibitors andmethods of screening with these compounds.

BACKGROUND

NF-κB is an ubiquitously expressed transcription factor that controlsthe expression of a diverse range of genes involved in inflammation,immune response, lymphoid differentiation, growth control anddevelopment. NF-κB resides in the cytoplasm as an inactive dimerconsisting of p50 and p65 subunits, bound to an inhibitory protein knownas IκB. The latter becomes phosphorylated and degraded in response tovarious environmental stimuli, such as pro-inflammatory cytokines,viruses, lipopolysaccharides, oxidants, UV light and ionizing radiation.This allows NF-κB to translocate to the nucleus where it activates genesthat play a key role in the regulation of inflammatory and immuneresponses, including genes that encode pro-inflammatory cytokines(IL-1β, TNF, GM-CSF, IL-2, IL-6, IL-11, IL-17), chemokines (IL-8,RANTES, MIP-1α, MCP-2), enzymes that generate mediators of inflammation(NO synthetase, cyclo-oxygenase), immune receptors (IL-2 receptor) andadhesion molecules (ICAM-1, VCAM-1, E-selectin). Some of these inducedproteins can in turn activate NF-κB, leading to the furtheramplification and perpetuation of the inflammatory response. Recently,NF-κB has been shown to have an anti-apoptotic role in certain celltypes, most likely by inducing the expression of anti-apoptotic genes.This function may protect tumor cells against anti-cancer treatments andopens the possibility to use NF-κB inhibiting compounds to sensitize thetumor cells and to improve the efficiency of the anti-cancer treatment.

Because of its direct role in regulating responses to inflammatorycytokines and endotoxin, activation of NF-κB plays an important role inthe development of different diseases such as (Barnes and Karin, 1997):chronic inflammatory diseases, i.e., rheumatoid arthritis, asthma andinflammatory bowel disease (Brand et al., 1996); acute diseases, i.e.,septic shock (Remick, 1995); Alzheimer's disease where the β-amyloidprotein activates NF-κB (Behl et al., 1997); atherosclerosis, whereNF-κB may be activated by oxidized lipids (Brand et al., 1997);autoimmune diseases, i.e., such as systemic lupus erythematosis(Kaltschmidt et al., 1994); or cancer by up-regulating certain oncogenesor by preventing apoptosis (Luque et al., 1997). In addition, NF-κB isalso involved in viral infection since it is activated by differentviral proteins, such as occurs upon infection with rhinovirus, influenzavirus, Epstein-Barr virus, HTLV, cytomegalovirus or adenovirus.Furthermore, several viruses such as HIV have NF-κB binding sites intheir promoter/enhancer regions (Mosialos, 1997).

Because of the potential role of NF-κB in many of the above mentioneddiseases, NF-κB and its regulators have drawn much interest as targetsfor the treatment of NF-κB related diseases. Glucocorticoids areeffective inhibitors of NF-κB, but they have endocrine and metabolicside effects when given systematically (Barnes et al., 1993).Antioxidants may represent another class of NF-κB inhibitors, butcurrently available antioxidants, such as acetyl-cysteine are relativelyweak and unspecific (Schreck et al., 1991). Aspirin and sodiumsalicylate also inhibit activation of NF-κB, but only at relatively highconcentrations (Kopp and Gosh, 1994). There are some natural inhibitorsof NF-κB such as glyotoxin, derived from Aspergillus, but thesecompounds are too toxic to be used as a drug (Pahl et al., 1996).Finally, there maybe endogenous inhibitors of NF-κB, such as IL-10, thatblocks NF-κB through an effect on IκB (Wang et al., 1995). However,total inhibition of NF-κB in all cell types for prolonged periods isunwanted, because NF-κB plays a crucial role in the immune response andother defensive responses.

An important role in the induction of NF-κB by TNF and IL1 has recentlybeen demonstrated for TNF-receptor associated factors, TRAF2 and TRAF6,which are recruited to the stimulated TNF-receptor and IL-1 receptor,respectively (Rothe et al., 1995; Cao et al., 1996). Over expression ofTRAF2 or TRAF6 activates NF-κB, whereas dominant negative mutantsinhibit TNF or IL-1 induced activation of NF-κB in most cell types.TRAF2 knock out studies have recently shown that TRAF2 is not absolutelyrequired for NF-κB activation, presumably because of redundancy withinthe TRAF family (Yeh et al. 1997). The TRAF induced signaling pathway toNF-κB was further resolved by the identification of the TRAF-interactingprotein NIK, which mediates NF-κB activation upon TNF and IL-1stimulation by association and activation of IκB kinase-α and β (IKK)(Malinin et al, 1997; Regnier et al., 1997; DiDonato et al., 1997; Zandiet al., 1997; Woronicz et al., 1997). The latter are part of a largemulti-protein NF-κB activation complex and are responsible forphosphorylation of IκB, leading to its subsequent degradation and totranslocation of released, active NF-κB to the nucleus. This allows amore specific inhibition of NF-κB activation by stimuli (including TNFand IL-1) that activate TRAF pathways. Based on this principle, WO97/37016 discloses the use of NIK and other TRAF interacting proteinsfor the modulation of NF-κB activity.

Another protein that can associate with TRAF2 is the zinc finger proteinA20 (Song et al., 1996). The latter is encoded by an immediate earlyresponse gene induced in different cell lines upon stimulation by TNF orIL-1 (Dixit et al, 1990). Interestingly, over expression of A20 blocksboth TNF and IL-1 induced NF-κB activation (Jaattela et al., 1996).However, the mechanism by which A20 blocks NF-κB activation is totallyunknown. In contrast to NIK, A20 does not seem to act directly on IκBresulting in alternative pathway to modulate NF-κB activation.

De Valck et al. (1997) isolated an A20 binding protein, so-called14-3-3, using a yeast two-hybrid assay and demonstrated that NF-κBinhibition was independent from the binding of A20 to 14-3-3.

SUMMARY OF THE INVENTION

It is shown herein that other new A20 interacting proteins unexpectedlycan modulate and/or inhibit NF-κB activation.

The invention includes an isolated functional protein comprising anamino acid sequence with 70-100% homology to the amino acid sequencedepicted in SEQ ID NO:2, or comprising an amino acid sequence with70-100% homology to the amino acid sequence depicted in SEQ ID NO:19,or, in the alternative, comprising an amino acid sequence with 70-100%homology to the amino acid sequence depicted in SEQ ID NO:6.

More specifically, the functional protein comprises an amino acidsequence with 70-100% homology to the amino acids 54-647 of SEQ ID NO:2,even more specifically the functional protein comprises an amino acidsequence with 70-100% homology to the amino acids 390-647 of SEQ IDNO:2, and/or comprising an amino acid sequence with 70-100% homology tothe amino acids 420-647 of SEQ ID NO:2.

Homology, in this context, means identical or similar to the referencedsequence, while known replacements/modifications of any of the aminoacids provided, are included as well. A homology search in this respectcan be performed with the BLAST-P (Basic Local Alignment Search Tool)program well known to a person skilled in the art. For the correspondingnucleic acid sequence homology is referred to the BLASTX and BLASTNprograms known in the art.

One aspect of the invention is to offer novel modulators and/orinhibitors of TNF and/or IL-1 induced NF-κB activation pathways.

An important embodiment of the invention is a protein comprising atleast the amino acids of SEQ ID NO:2.

Another embodiment of the invention is a protein comprising at least theamino acids 54-647 of SEQ ID NO:2, as represented in SEQ ID NO:19.

A further embodiment of the invention is a protein comprising at leastthe amino acids of SEQ ID NO:6.

A further aspect of the invention is the use of a protein comprising theamino acids 420-647 of SEQ ID NO:2 to modulate and/or inhibit the NF-κBrelated pathway, especially the TNF and/or IL-1 induced pathways.

In addition, the invention concerns the use of a protein, comprising theconsensus sequence shown in SEQ ID NO:8 and/or SEQ ID NO:9, to modulateand/or inhibit the TNF and/or IL-1 induced, NF-κB related pathway.

Another aspect of the invention is the use of these proteins in ascreening method to screen compounds that interfere with the interactionof these protein(s) with other protein components of the NF-κB relatedpathway.

Another embodiment of the invention is the use of the above mentionedproteins, or the use of protein components screened by the abovementioned method to sensitize tumor cells and/or improve the anti-cancertreatment.

The present invention also relates to a method for identifying andobtaining an activator or inhibitor of A20 interacting protein(s)comprising the steps of:

-   -   (a) combining a compound to be screened with a reaction mixture        containing the protein of the invention and a read out system        capable of interacting with the protein under suitable        conditions;    -   (b) maintaining the reaction mixture in the presence of the        compound or a sample comprising a plurality of compounds under        conditions which permit interaction of the protein with the read        out system;    -   (c) identifying or verifying a sample and compound,        respectively, which leads to suppression or activation of the        read out system.

The term “read out system” in context with the present invention means aDNA sequence which upon transcription and/or expression in a cell,tissue or organism provides for a scorable and/or selectable phenotype.Such read out systems are well known to those skilled in the art andcomprise, for example, recombinant DNA molecules and marker genes asdescribed above.

The term “plurality of compounds” in a method of the invention isunderstood as a plurality of substances which may be identical.

The compound or plurality of compounds may be comprised in, for example,samples, e.g., cell extracts from animals or microorganisms.Furthermore, the compound(s) may be known in the art but hitherto notknown to be capable of suppressing or activating A20 interactingproteins. The reaction mixture may be a cell free extract or maycomprise a cell or tissue culture. Suitable set ups for the method ofthe invention are known to the person skilled in the art and are, forexample, generally described in Alberts et al., Molecular Biology of theCell, (3rd ed. 1994). The plurality of compounds may be, for instance,added to the reaction mixture or culture medium, or may be injected intothe cell.

If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is possible toisolate the compound from the original sample identified as containingthe compound capable of suppressing or activating A20 interactingproteins. Additionally, one can further subdivide the original sample,for example, if it consists of a plurality of different compounds, so asto reduce the number of different substances per sample. The method canthen be repeated with the subdivisions of the original sample. Dependingon the complexity of the samples, the steps described above can beperformed several times, preferably until the sample identifiedaccording to the method of the invention only comprises a limited numberof, or only one substance(s). Preferably, the sample comprisessubstances of similar chemical and/or physical properties, and mostpreferably the substances are identical. The compounds which can betested and identified according to a method of the invention may beexpression libraries, e.g., cDNA expression libraries, peptides,proteins, nucleic acids, antibodies, small organic compounds, hormones,peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995),879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198and references cited supra).

The invention also relates to a DNA sequence encoding the referencedproteins or a DNA sequence encoding an immunologically active and/orfunctional fragment of such a protein, selected from the groupconsisting of:

-   -   (a) DNA sequences comprising a nucleotide sequence encoding a        protein comprising the amino acid sequence as given in SEQ ID        NO:2;    -   (b) DNA sequences comprising a nucleotide sequence as given in        SEQ ID NO: 1;    -   (c) DNA sequences hybridizing with the complementary strand of a        DNA sequence as defined in (a) or (b) and encoding an amino acid        sequence which is at least 70% identical to the amino acid        sequence encoded by the DNA sequence of (a) or (b);    -   (d) DNA sequences, the nucleotide sequence of which is        degenerated as a result of the genetic code to a nucleotide        sequence of a DNA sequence as defined in any one of (a) to (c);        and    -   (e) DNA sequences encoding a fragment of a protein encoded by a        DNA sequence of any one of (a) to (d).

Thus, the invention consists of DNA molecules, also called nucleic acidsequences, encoded for the above mentioned proteins preferably a nucleicacid sequence, with 70-100% homology to the DNA sequence depicted in SEQID NO:1, and/or a nucleic acid sequence with 70-100% homology to the DNAsequence depicted in SEQ ID NO:5.

Homology in this context means that the respective nucleic acidmolecules or encoded proteins are functionally and/or structurallyequivalent. The nucleic acid molecules that are homologous to thenucleic acid molecules described above and that are derivatives of thenucleic acid molecules are, for example, variations of the nucleic acidmolecules which represent modifications having the same biologicalfunction. In particular, the modifications encode proteins with the sameor substantially the same biological function. They may be naturallyoccurring variations, such as sequences from other varieties or species,or mutations. These mutations may occur naturally or may be obtained bymutagenesis techniques. The allelic variations may be naturallyoccurring allelic variants as well as synthetically produced orgenetically engineered variants.

The proteins encoded by the various derivatives and variants of theabove-described nucleic acid molecules have similar commoncharacteristics, such as biological activity, molecular weight,immunological reactivity, conformation, etc., as well as physicalproperties, such as electrophoretic mobility, chromatographic behavior,sedimentation coefficients, pH optimum, temperature optimum, stability,solubility, spectroscopic properties, etc.

The present invention also relates to vectors, particularly plasmids,cosmids, viruses, bacteriophages and other vectors used conventionallyin genetic engineering that contain a nucleic acid molecule according tothe invention. Methods which are well known to those skilled in the artcan be used to construct various plasmids and vectors; see, for example,the techniques described in Sambrook, Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory (1989) N.Y.

Alternatively, the nucleic acid molecules and vectors of the inventioncan be reconstituted into liposomes for delivery to target cells.

In a preferred embodiment, the nucleic acid molecule present in thevector is operably linked to (a) control sequence(s) which allow theexpression of the nucleic acid molecule in prokaryotic and/or eukaryoticcells.

The term “control sequence” refers to regulatory DNA sequences which arenecessary to affect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism. In procaryotes, control sequences generally includepromoter, ribosomal binding site, and terminators. In eucaryotes,control sequences generally include promoters, terminators and, in someinstances, enhancers, transactivators or transcription factors. The term“control sequence” is intended to include, at a minimum, all componentsthat are necessary for expression, and may also include additionaladvantageous components.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences. In case the control sequence is a promoter, it is known to askilled person that double-stranded nucleic acid is used.

Thus, the vector of the invention is preferably an expression vector. An“expression vector” is a construct that can be used to transform aselected host cell and provides for expression of a coding sequence inthe selected host. Expression vectors can, for instance, be cloningvectors, binary vectors or integrating vectors. Expression comprisestranscription of the nucleic acid molecule preferably into atranslatable mRNA. Regulatory elements ensuring expression inprokaryotic and/or eukaryotic cells are well known to those skilled inthe art.

The present invention furthermore relates to host cells comprising avector as described herein or a nucleic acid molecule according to theinvention wherein the nucleic acid molecule is foreign to the host cell.

By “foreign” is meant that the nucleic acid molecule is eitherheterologous or homologous with respect to the host cell. “Heterologous”means derived from a cell or organism with a different genomicbackground. “Homologous” means located in a different genomicenvironment than the naturally occurring counterpart of the nucleic acidmolecule. Thus, if the nucleic acid molecule is homologous with respectto the host cell, it is not located in its natural location in thegenome of the host cell, but it is surrounded by different genes. Inthis case, the nucleic acid molecule may be either under the control ofits own promoter or under the control of a heterologous promoter. Thevector or nucleic acid molecule, according to the invention, which ispresent in the host cell may either be integrated into the genome of thehost cell or it may be maintained in some form extra-chromosomally. Itis also possible that the nucleic acid molecule of the invention can beused to restore or create a mutant gene via homologous recombination(Paszkowski (ed.), Homologous Recombination and Gene Silencing inPlants, (Kluwer Academic Publishers 1994)).

The host cell can be any prokaryotic or eukaryotic cell, such asbacterial, insect, fungal, plant or animal cells. Preferred fungal cellsare, for example, those of the genus Saccharomyces, in particular thoseof the species S. cerevisiae.

The invention also includes a method for preparing A20 interactingproteins which method comprises the cultivation of host cells that dueto the presence of a vector or a nucleic acid molecule according to theinvention, are able to express such a protein under conditions whichallow expression of the protein and thus recovery of the so-producedprotein from the culture.

The term “expression” means the production of a protein or nucleotidesequence in the cell. However, the term also includes expression of theprotein in a cell-free system. It includes transcription into an RNAproduct, post-transcriptional modification and/or translation to aprotein product or polypeptide from a DNA encoding that product, as wellas possible post-translational modifications. Depending on the specificconstructs and conditions used, the protein may be recovered from thecells, from the culture medium or from both. For the person skilled inthe art, it is well known that it is not only possible to express anative protein, but also to express the protein as fusion polypeptidesor to add signal sequences directing the protein to specificcompartments of the host cell, for example, ensuring secretion of thepeptide into the culture medium, etc. Furthermore, such a protein andfragments thereof can be chemically synthesized and/or modifiedaccording to standard methods.

The terms “protein” and “polypeptide” used in this application areinterchangeable. “Polypeptide” refers to a polymer of amino acids (aminoacid sequence) and does not refer to a specific length of the molecule.Thus peptides and oligopeptides are included within the definition ofpolypeptide. This term also refers to and includes post-translationalmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),polypeptides with substituted linkages, as well as other modificationsknown in the art, both naturally occurring and non-naturally occurring.

The present invention furthermore relates to proteins encoded by thenucleic acid molecules according to the invention or produced orobtained by the methods described herein, and to functional and/orimmunologically active fragments of such A20 interacting proteins. Theproteins and polypeptides of the present invention are not necessarilytranslated from a designated nucleic acid sequence. The polypeptides maybe generated in any manner, including for example, chemical synthesis,or expression of a recombinant expression system, or isolation from asuitable viral system. The polypeptides may include one or more analogsof amino acids, phosphorylated amino acids or unnatural amino acids.Methods of inserting analogs of amino acids into a sequence are known inthe art. The polypeptides may also include one or more labels, which areknown to those skilled in the art. In this context, it is alsounderstood that the proteins according to the invention may be furthermodified by conventional methods known in the art. By providing theproteins according to the present invention it is also possible todetermine fragments which retain biological activity, namely the mature,processed form. This allows the construction of chimeric proteins andpeptides comprising an amino sequence derived from the protein of theinvention, which is crucial for its binding activity. The otherfunctional amino acid sequences may be either physically linked by, forexample, chemical means to the proteins of the invention or may be fusedby recombinant DNA techniques well known in the art.

The term “functional fragment of a sequence” or “functional part of asequence” means a truncated sequence of the original sequence referredto. The truncated sequence (nucleic acid or protein sequence) can varywidely in length; the minimum size being a sequence of sufficient sizeto provide a sequence with at least a comparable function and/oractivity of the original sequence referred to, while the maximum size isnot critical. In some applications, the maximum size usually is notsubstantially greater than that required to provide the desired activityand/or function(s) of the original sequence. Typically, the truncatedamino acid sequence will range from about 5 to about 60 amino acids inlength. More typically, however, the sequence will be a maximum of about50 amino acids in length, preferably a maximum of about 30 amino acids.It is desirable to select sequences of at least about 10, 12 or 15 aminoacids, up to a maximum of about 20 or 25 amino acids.

Furthermore, folding simulations and computer redesign of structuralmotifs of the protein of the invention can be performed usingappropriate computer programs (Olszewski, Proteins 25 (1996), 286-299;Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679). Computer modeling ofprotein folding can be used for the conformational and energeticanalysis of detailed peptide and protein models (Monge, J. Mol. Biol.247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45).In particular, the appropriate programs can be used for theidentification of interactive sites of the inventive protein , itsreceptor, its ligand or other interacting proteins by computer assistantsearches for complementary peptide sequences (Fassina, Immunomethods 5(1994), 114-120. Further appropriate computer systems for the design ofprotein and peptides are described in the prior art, for example inBerry, Biochem. Soc. Trans. 22 (1994),1033-1036; Wodak, Ann. N. Y. Acad.Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. Theresults obtained from the above-described computer analysis can be usedfor, e.g., the preparation of peptidomimetics of the protein of theinvention or fragments thereof. Such pseudopeptide analogues of thenatural amino acid sequence of the protein may very efficiently mimicthe parent protein (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224).For example, incorporation of easily available achiral amino acidresidues into a protein of the invention or a fragment thereof resultsin the substitution of amide bonds by polymethylene units of analiphatic chain, thereby providing a convenient strategy forconstructing a peptidomimetic (Banerjee, Biopolymers 39 (1996),769-777). Superactive peptidomimetic analogues of small peptide hormonesin other systems are described in the prior art (Zhang, Biochem.Biophys. Res. Commun. 224 (1996), 327-331). Appropriate peptidomimeticsof the protein of the present invention can also be identified by thesynthesis of peptidomimetic combinatorial libraries through successiveamide alkylation and testing the resulting compounds, e.g., for theirbinding and immunological properties. Methods for the generation and useof peptidomimetic combinatorial libraries are described in the priorart. See, e.g., Ostresh, Methods in Enzymology 267 (1996), 220-234;Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.

Furthermore, a three-dimensional and/or crystallographic structure ofthe protein of the invention can be used for the design ofpeptidomimetic inhibitors of the biological activity of the protein ofthe invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber,Bioorg. Med. Chem. 4 (1996), 1545-1558).

Furthermore, the present invention relates to antibodies specificallyrecognizing a A20 interacting protein according to the invention orparts, i.e. specific fragments or epitopes, of such a protein. Theantibodies of the invention can be used to identify and isolate otherA20 interacting proteins and genes in any organism. These antibodies canbe monoclonal antibodies, polyclonal antibodies or synthetic antibodiesas well as fragments of antibodies, such as Fab, Fv or scFv fragmentsetc. Monoclonal antibodies can be prepared, for example, by thetechniques as originally described in Kohler and Milstein, Nature 256(1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise thefusion of mouse myeloma cells to spleen cells derived from immunizedmammals. Furthermore, antibodies or fragments thereof to theaforementioned peptides can be obtained by using methods which aredescribed, for example, in Harlow and Lane “Antibodies, A LaboratoryManual”, CSH Press, Cold Spring Harbor, 1988. These antibodies can beused, for example, for the immunoprecipitation and immunolocalization ofproteins according to the invention. Additionally, the antibodies can beused for the monitoring of the synthesis of such proteins, for example,in recombinant organisms, and for the identification of compoundsinteracting with the protein according to the invention. For example,surface plasmon resonance as employed in the BIAcore system can be usedto increase the efficiency of phage antibodies selections, yielding ahigh increment of affinity from a single library of phage antibodieswhich bind to an epitope of the protein of the invention (Schier, HumanAntibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods183 (1995), 7-13). In many cases, the binding phenomena of antibodies toantigens is equivalent to other ligand/anti-ligand binding.

The invention also relates to a diagnostic composition comprising atleast one of the nucleic acid molecules, vectors, proteins, antibodiesor compounds described herein and, optionally, a suitable means fordetection.

The diagnostic compositions may be used in methods for detectingexpression of related A20 interacting proteins. This is accomplished bydetecting the presence of the corresponding mRNA which comprisesisolation of mRNA from a cell, contacting the mRNA obtained with a probecomprising a nucleic acid probe as described herein under hybridizingconditions, detecting the presence of mRNA hybridized to the probe,and/or detecting the expression of the protein in the cell. Furthermethods of detecting the presence of a protein according to the presentinvention comprises immunotechniques well known in the art, for example,enzyme-linked immunosorbent assays.

The invention also relates to a pharmaceutical composition comprisingone or more compounds, obtained by a screening method described herein,in a biologically active amount, for the treatment of NF-κB relateddiseases such as respiratory disorders, particularly adult respiratorydistress syndrome, allograft rejection, chronic inflammatory diseasessuch as rheumatoid arthritis, asthma or inflammatory bowel disease,and/or autoimmune diseases such as systemic lupus erythematosis.

In another aspect, the invention relates to a pharmaceutical compositioncomprising one or more of the A20 interacting proteins in a biologicallyactive amount, for the treatment of NF-κB related diseases such asrespiratory disorders, particularly adult respiratory distress syndrome,allograft rejection, chronic inflammatory diseases such as rheumatoidarthritis, asthma or inflammatory bowel disease, and/or autoimmunediseases such as systemic lupus erythematosis.

The invention also concerns a pharmaceutical composition comprising oneor more of the A20 interacting proteins and/or one or more of the abovementioned compounds in a biologically active amount, for a treatment tosensitize tumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Tissue distribution of ABIN transcripts. A Northern blot ofpoly(A)+RNA (2 μg per lane) of various murine tissues (Clontech) wasprobed with the fragment of ABIN cloned by two hybrid analysis coveringthe C-terminal sequences ABIN (390-599). RNA size markers are indicatedin kB. Expression of β-actin served as a control for the quantity of RNAloaded.

FIG. 2: Co-immunoprecipitation of A20 and ABIN after transienttransfection of the encoding plasmids of E-tagged ABIN and GreenFluorescent Protein (GFP), GFP-A20, GFP-A20(369-775), GFP-A20(1-368) oran empty expression vector as a negative control in 239T cells.Immunoprecipitation (upper panel) was performed with anti-GFP antibodyand Western blot detection with anti E-tag antibody. To controlexpression levels of ABIN, 10 μl aliquots of lysates were separated bysodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)and Western blot detection with anti-E-tag antibody (lower panel).

FIG. 3: Co-immunoprecipitation of the C-terminal fragment of ABINlacking the putative leucine zipper structure with GFP-A20 upontransient over expression in 293T cells. Immunoprecipitation andexpression levels were detected as described for full length ABIN andare shown in the upper and lower panel, respectively.

FIG. 4 is a bar graph depicting the effect of ABIN or fragments of ABINon the TNF- and IL-1-induced activation of NF-κB, as measured byreporter gene activity. 293T cells were transiently transfected with 100ng pUT651, 100 ng pNFconluc and 100 ng expression plasmid and stimulatedwith hTNF (1000 IU/ml) or mIL1β (20 ng/ml) during 6 hours. As a control,100 ng of plasmids encoding GFP or GFP-A20 were transfected.

FIG. 5 is a bar graph depicting the effect of transient transfection ofsub-optimal quantities of expression plasmids encoding A20 (5 ng) andABIN (20 ng) on TNF mediated NF-κB induction in 293T cells. In bothexperiments, standard deviations were less than 10%.

FIG. 6 is a bar graph depicting the effect of ABIN or fragments of ABINon the TPA-induced activation of NF-κB, as measured by reporter geneactivity. 293T cells were transiently transfected with 100 ng pUT651,100 ng pNFconluc and 100 ng expression plasmid and stimulated with TPA(200 ng/ml) during 6 hours. As a control, 100 ng of plasmids encodingGFP or GFP-A20 were transfected.

FIG. 7 is a bar graph depicting the effect of ABIN2 on the TNF- andTPA-induced activation of NF-κB, as measured by reporter gene activity.293T cells were transiently transfected with 100 ng pUT651, 100 ngpNFconluc and 600 ng expression plasmid and stimulated with hTNF (1000IU/ml) or TPA (200 ng/ml) during 6 hours. As a control, 600 ng ofplasmid encoding GFP or GFP-A20 was transfected.

FIG. 8 is a bar graph depicting the effect of full length ABIN on NF-κBactivation in 293T cells induced by over expression of TRADD, RIP,TRAF2, NIK or p65 after transfection of 300 ng of their encodingplasmids, together with 100 ng pUT651, 100 ng pNFconluc and 500 ngpCAGGS-ABIN. Cells were lysed 24 hours after transfection, andluciferase and β-galactosidase activity were measured.

FIG. 9 specifically is a bar graph depicting the effect of truncatedABIN containing the leucine zipper structure (ABIN(390-647)) on TRADD,RIP, TRAF2 or NIK induced NF-κB activation. In both experiments,standard deviations were less than 10%.

FIGS. 10, 11 and 12: As an overview, these figures illustrate the effectof site specific mutations in two regions of ABIN on its binding withA20 and on its inhibition of NF-κB activation.

FIG. 10 shows the co-immunoprecipitation of mutated ABIN with A20 aftertransient expression of these genes in 293T cells. Cells weretransfected with the plasmids pCAGGS-GFP or pCAGGS-GFP/A20 together withthe plasmids encoding ABIN or its site specific mutants (ABIN-MUT1,ABIN-MUT2, ABIN-MUT3 or ABIN-MUT4). Lysates of these cells wereimmunoprecipitated with polyclonal anti-GFP antibody and separated onSDS-PAGE. Western blot analysis was performed with monoclonal anti-E-tagantibody to look for the Co-immunoprecipitation of ABIN or its mutants(upper panel). Lower panels show total expression levels of GFP, GFP/A20and ABIN. In this case, a fraction of the total lysate was separated bySDS-PAGE and expression was detected with anti-GFP or anti-E-tagantibodies.

FIG. 11 is a bar graph depicting the effect of mutated ABIN onTNF-induced NF-κB activation. 293T cells were transiently transfectedwith 100 ng pUT651, 100 ng pNFconluc and 200 ng expression plasmid asindicated and stimulated with TNF (1000 IU/ml) during 6 hours. Cellextracts were analyzed for luciferase and β-galactosidase activity andplotted as luc/gal, which is representative for NF-κB activity. Eachvalue is the mean (N=3) with standard deviations less than 10%.

FIG. 12 is a bar graph depicting the dominant negative effect ofABIN-MUT2, ABIN-MUT3 and ABIN-MUT4 on the NF-κB inhibiting function ofABIN. 293T cells were transiently transfected with 100 ng pUT651, 100 ngpNFconluc and 200 ng pCAGGS-ABIN or empty vector. In addition, 600 ng ofthe expression vectors encoding ABIN-MUT2, ABIN-MUT3, ABIN-MUT4 or emptyvector were co-transfected, as indicated. Cells were stimulated with TNF(1000 IU/ml) during 6 hours. Cell extracts were analyzed for luciferaseand β-galactosidase activity and plotted as 1 uc/gal.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided in order to further illustrateand define the meaning and scope of the various terms used in thecurrent description.

The term “treatment”, “treating” or “treat” means any treatment of adisease in a mammal, including: (1) preventing the disease causing theclinical symptoms of the disease not to develop; (2) inhibiting thedisease arresting the development of the clinical symptoms; and/or (3)relieving the disease causing the regression of clinical symptoms.

The term “effective amount” means a dosage sufficient to providetreatment for the disease state being treated. This will vary dependingon the patient, the disease and the treatment being effected.

“Capable of interacting” means that a protein can form a complex withanother protein, as can be measured using a yeast two hybrid system, orwith co-immunoprecipitation, or with equivalent systems known to peopleskilled in the art.

“Functional” protein or fragment means a protein or fragment that iscapable to interact with the zinc finger protein A20, or with anotherprotein of the NF-κB related pathway.

“Protein A20” (“A20”) means the TNF induced zinc finger protein,described by (Dixit et al., 1990; Opipari et al., 1990; Tewari et al.,1995), or an active fragment thereof, such as the zinc finger containingpart (amino acids 387-790 of human A20; amino acids 369-775 of murineA20).

The terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”,“nucleotide sequence”, “DNA sequence” or “nucleic acid molecule(s)” asused herein refer to a polymeric form of nucleotides of any length,either ribonucleotides or deoxyribonucleotides. These terms refer onlyto the primary structure of the molecule. Thus, the term includesdouble- and single-stranded DNA, and RNA. It also includes known typesof modifications, for example, methylation, “caps” substitution of oneor more of the naturally occurring nucleotides with an analog.Preferably, the DNA sequence of the invention comprises a codingsequence encoding the above defined A20 interacting protein.

A “coding sequence” is a nucleotide sequence which is transcribed intomRNA and/or translated into a polypeptide when placed under the controlof appropriate regulatory sequences. The boundaries of the codingsequence are determined by a translation start codon at the 5′-terminusand a translation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to mRNA, cDNA, recombinant nucleotidesequences or genomic DNA, while introns may be present as well undercertain circumstances.

“Consensus sequence” means a stretch of at least 15 amino acids, showing50-100% homology, preferably between 70-100% homology between ABIN andABIN2.

“Compound” means any chemical or biological compound, including simpleor complex inorganic or organic molecules, peptides, peptido-mimetics,proteins, antibodies, carbohydrates or nucleic acids, that interfere inthe binding between a protein depicted in SEQ ID NOS:2, 19, 6, 8 or 9with a compound of the NF-κB related pathway, such as A20.

As used herein, the term “composition” refers to any composition such asa pharmaceutical composition comprising an active ingredient of anisolated functional protein according to the present invention. This maybe performed in the presence of suitable excipients known to the skilledman and may be administered in the form of any suitable composition asdetailed below, and by any suitable method of administration within theknowledge of a skilled man. The preferred route of administration isparenterally. In parenteral administration, the compositions of thisinvention will be formulated in a unit dosage injectable form such as asolution, suspension or emulsion, in association with a pharmaceuticallyacceptable excipient. Such excipients are inherently nontoxic andnon-therapeutic. Examples of such excipients are saline, Ringer'ssolution, dextrose solution and Hank's solution. Non-aqueous excipientssuch as fixed oils and ethyl oleate may also be used. A preferredexcipient is 5% dextrose in saline. The excipient may contain minoramounts of additives such as substances that enhance isotonicity andchemical stability, including buffers and preservatives.

The isolated functional protein of the invention is administered at aconcentration that is therapeutically effective to prevent allograftrejection, GVHD, allergy and autoimmune diseases. The dosage and mode ofadministration will depend on the individual. Generally, thecompositions are administered so that the isolated functional protein isgiven at a dose between 1 μg/kg and 10 mg/kg, more preferably between 10μg/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg. Preferably,it is given as a bolus dose. Continuous short time infusion (during 30minutes) may also be used. The compositions comprising the isolatedfunctional protein according to the invention may be infused at a dosebetween 5 and 20 μg/kg/minute, more preferably between 7 and 15μg/kg/minute.

The “therapeutically effective amount” of the isolated functionalprotein needed in a specific case, according to the invention, should bedetermined as being the amount sufficient to cure the patient in need oftreatment or at least to partially arrest the disease and itscomplications. Amounts effective for such use will depend on theseverity of the disease and the general state of the patient's health.Single or multiple administrations may be required depending on thedosage and frequency as required and tolerated by the patient.

With regard to the use of the isolated functional protein of the presentinvention to prevent allograft rejection, it should be stressed that theproteins of the present invention or the compositions comprising thesame may be administered before, during or after the organtransplantation as is desired from case to case. In case the protein orthe compositions comprising the same are administered directly to thehost, treatment will preferably start at the time of the transplantationand continue afterwards in order to prevent the activation anddifferentiation of host T cells against the MHC on the allograft. Incase the donor organ is ex vivo perfused with the functional proteinaccording to the invention or the compositions comprising the same,treatment of the donor organ ex vivo will start before the time of thetransplantation of the donor organ in order to prevent the activationand differentiation of host T cells against the MHC on the allograft.

The invention is hereunder further explained by way of examples withoutbeing restrictive in the scope of the current invention.

EXAMPLES Example 1 Isolation of the Novel Inhibitors

The novel inhibitors of the NF-κB pathway were isolated using a yeasttwo-hybrid assay, with protein A20 as bait. The yeast two-hybrid assaywas purchased from Clontech Laboratories (Palo Alto, Calif.). Thescreening of an L929r2 cDNA library with pAS2-A20 was describedpreviously (De Valck et al., 1997). Yeast colonies expressinginteracting proteins were selected by growth on minimal media lackingTryptophan, Leucine and Histidine, in the presence of 5 mM3-amino-1,2,4-triazole and by screening for β-gal activity. Plasmid DNAwas extracted from the positive colonies and the pGAD424 vectorsencoding candidate A20 interacting proteins were recovered byelectroporation in the E. coli strain HB101 and growth on media lackingLeucine.

From 1.3×10⁶ transformants, 11 clones expressed A20 interactingproteins, including A20 itself (De Valck et al., 1996) and 14-3-3proteins (De Valck et al., 1997). Three clones contained C-terminalfragments of the same cDNA encoding an unknown protein that herewith isnamed A20 Binding Inhibitor of NF-κB activation (ABIN) and 1 clonecontained the C-terminal fragment (1136 bp) of an unknown protein thatherewith is called ABIN2.

Full length ABIN cDNA was subsequently isolated from the L929r2 cDNAlibrary by colony hybridization (De Valck et al.,1996) with an ABINfragment (corresponding to amino acids 390-599) cloned by two-hybridanalysis as a probe. Several cDNA's were isolated and in the longestcDNA stop codons were identified in all three reading frames 5′ of apotential initiator methionine. Two different splice variants were foundof approximately 2800 and 2600 nucleotides long, with an open readingframe of 1941 and 1781 nucleotides respectively, initiating at twodifferent methionines (ABIN (1-647) (SEQ ID NO:2) and ABIN (54-647) (SEQID NO:19)). These cDNA's encode proteins of 72 and 68 kDa containing anamphipathic helix with 4 consecutive repeats of a leucine followed by 6random amino acids residues characteristic of a leucine zipperstructure.

Full length cDNA of ABIN2 was isolated from murine heart by 5′ RACE(SMART PCR cDNA synthesis kit, Clontech), using a 3′ primer hybridizingto an EST clone (572231) which corresponds to the ABIN2 fragmentisolated by two hybrid analysis, but with 507 extra nucleotides at the5′ end. A 1,967 nucleotide long cDNA was isolated (SEQ ID NO:5), with anopen reading frame of 1,290 nucleotides long, encoding a protein of 430amino acids (SEQ ID NO:6).

Example 2 Expression Pattern of ABIN and ABIN2.

Northern blot analysis revealed that both ABIN and ABIN2 are expressedin all murine tissues tested (heart, brain, spleen, lung, liver,skeletal muscle, kidney, testis: see FIG. 1; only the data for ABIN areshown). ABIN is present as mRNA of approximately 2800 bp which is inaccordance with the length of the cloned full length cDNA. In contrastto A20, ABIN mRNA is constitutively expressed in both TNF-sensitive andTNF-resistant subclones derived from the parental cell line L929s,irrespective of TNF stimulation.

ABIN2 is present as mRNA of approximately 2,000 bp, which is inaccordance with the length of the cloned, full length cDNA.

Example 3 Study of the Interaction of the ABIN and ABIN2 Proteins andProtein Fragments With A20.

Full length ABIN(1-647) and ABIN(54-647) were able to bind A20 in ayeast two hybrid assay, confirming the original interaction found withthe 3 C-terminal fragments ABIN(390-599), ABIN(249-647) andABIN(312-647). The latter contain the putative leucine zipper proteininteraction motif (397-420).

Further analysis was carried out by co-immunoprecipitation. Theeukaryotic plasmids for ABIN and its fragments as well as for ABIN2 weremade by inserting the corresponding PCR fragment in frame with anN-terminal E-tag into the mammalian expression plasmid pCAGGS (Niwa etal., 1991). cDNA encoding mutant GFP(S65T) and a fusion protein ofGFP(S65T) with murine A20 were also cloned in pCAGGS.

2×10⁶ human embryonic kidney 293T cells were plated on 10-cm Petridishes and transiently transfected with the suitable plasmids by calciumphosphate-DNA co-precipitation. 24 hours after transfection, cells werelysed in 500 μl of lysis buffer (50 mM Hepes pH 7.6, 250 mM NaCl, 0.1%Nonidet P-40 and 5 mM EDTA). Lysates were incubated with 5 μl of rabbitanti-GFP antibody (Clontech) and immunocomplexes were immobilized onprotein A-trisacryl (Pierce). The latter was washed twice with lysisbuffer and twice with lysis buffer containing 1 M NaCl. Co-precipitatingproteins were separated by SDS-PAGE and revealed by Western blottingwith mouse anti-E-tag antibody (Pharmacia).

Full length ABIN as well as the C-terminal fragment lacking the leucinezipper motif (ABIN(420-647)) was still able to co-immunoprecipitate withA20 in 293T cells that were transiently transfected with an expressionplasmid for chimeric GFP-A20 protein and full length or truncated ABINwith an N-terminal E-tag (FIG. 2). Interaction of ABIN with A20 requiredthe C-terminal, zinc finger containing part of A20 (A20(369-775)). Thisdomain was shown previously to be required for dimerization of A20 andfor the interaction of A20 with 14-3-3 protein (De Valck et al., 1996;De Valck et al., 1997). In contrast, the N-terminal part of human A20(A20(1-386)) was previously shown to interact with TRAF2 (Song et al.,1996), suggesting that A20 acts as an adapter protein between TRAF2 andABIN. The interaction between A20 and ABIN was not influenced bystimulation with TNF.

To characterize the sub-cellular distribution of ABIN, we transientlytransfected GFP-A20- and E-tagged ABIN cDNA in 293T cells and analyzedtheir expression by means of GFP fluorescence and immunofluorescence viathe anti-E tag antibody. 4×10⁵ 293T cells were seeded on cover slips in6-well plates and transfected with 1 μg plasmid DNA. 24 hours aftertransfection, cells were fixed on the cover slips with 3%paraformaldehyde. Upon permeabilization with 1% Triton X-100, the cellswere incubated for 2 hours with mouse anti-E-tag antibody (1/1000)followed by a second incubation with anti-mouse Ig antibody coupled tobiotin (Amersham, 1/1000). After subsequent incubation with streptavidincoupled to Texas Red (Amersham), fluorescence can be analyzed viafluorescence microscopy (Zeiss, Axiophot) using a filter set withexcitation at 543 nm and emission at 600 nm. In the same cells,fluorescence of GFP can be detected at a different wave length, namelyabsorption at 485 nm and emission at 510 nm.

ABIN co-localized with A20 throughout the cytoplasm, both inunstimulated and in TNF stimulated cells. This observation makes theexistence of regulatory redistribution events rather unlikely.

Example 4 Sequence Analysis of the cDNA's.

Nucleotide sequence analysis was carried out using cycle sequencing onan ABI373A sequencer (Applied Biosystems, Foster City, Calif.). Thesequence of full length ABIN is shown in SEQ ID NO:1; the sequence ofABIN2 is shown in SEQ ID NO:5.

Database similarity searches (BLAST) showed that ABIN is the murinehomologue of the partial human cDNA encoding a protein with an unknownfunction (Genbank accession number D30755; Nagase et al., 1995).Moreover, ABIN shows homology with a partial human immunodeficiencyvirus (HIV) Nef interacting protein, NIP40-1 (no called Naf1=nefassociated factor 1; Fukushi et al., FEBS Letters, 442 (1999), 83-88).HIV-Nef contributes substantially to disease pathogenesis by augmentingvirus replication and markedly perturbing T-cell function.Interestingly, the effect of Nef on host cell activation has beenexplained in part by its interaction with specific cellular proteinsinvolved in signal transduction (Harris, 1996) of which ABIN might be anexample.

There are no proteins in the database that are clearly homologous withABIN2. However, by comparing ABIN2 with ABIN, one can define twohomologous regions and derive two consensus sequences (SEQ ID NO:8 andSEQ ID NO:9)) that may be important for the interaction of theseproteins with A20, and/or for their further function in signaltransduction. A consensus sequence of ABIN and ABIN2 is found from aminoacids 423 to 441 and 475 to 495 of SEQ ID NO:2 for ABIN, and from aminoacids 256 to 274 and 300 to 320 of SEQ ID NO:6 for ABIN2. The consensussequence illustrates 22 amino acids from ABIN and ABIN2 that overlapwith one another.

Example 5

Role of ABIN, ABIN Fragments and/or ABIN2 in the TNF-, IL-1- and/orTPA-induced Transduction Pathway Leading to NF-κB Activation, asMeasured by Reporter Gene Activity.

The construction of the ABIN, ABIN-fragments and ABIN2 plasmids wascarried out as described above. The plasmid pNFconluc, encoding aluciferase reporter gene driven by a minimal NF-κB responsive promoterdescribed by Kimura et al. (1986) and the plasmid pUT651, encodingβ-galactosidase was obtained from Eurogentec (Seraing, Belgium). NF-κBactivity was determined by the NF-κB dependent expression of aluciferase reporter gene. Therefore, 293T cells were plated in 6-wellplates at 4×10⁵ cells per well and transiently transfected by thecalcium phosphate-DNA coprecipitation method. Each transfectioncontained 800 ng of the expression plasmids, as well as 100 ng ofpNFconluc plasmid as reporter and 100 ng pUT651 plasmid as a referencefor transfection efficiency. 24 hours after transfection, these cellswere trypsinized and seeded on a 24-well plate. Another 24 hours later,cells were either stimulated with 1000 IU/ml hTNF, 20 ng/ml mIL1-β, 200ng/ml TPA (Sigma) or left untreated. After 6 hours of stimulation, cellswere lysed in 200 μl lysis buffer and analyzed for luciferase andβ-galactosidase activity as described in De Valck et al., 1997. GFP andGFP-A20 served as negative and positive controls, respectively.

Similar to A20, both splice variants of ABIN were able to block TNF orIL-1 induced NF-κB activation in these cells, with the shorterN-terminal truncated isoform being slightly more effective (FIG. 4).Moreover, structure-function analysis of ABIN deletion mutants revealedthat the NF-κB inhibiting activity resides in the C-terminal 228 aminoacids (ABIN(420-647) SEQ ID NO:10) which are also sufficient forinteraction with A20. The latter ABIN-mutant no longer contains theleucine zipper structure, demonstrating that this protein domain is notinvolved in the interaction with A20 nor in the inhibition of NF-κB(FIG. 4). Overexpression of a combination of suboptimal doses of A20 andABIN, that on their own were not sufficient to inhibit NF-κB activation,diminished NF-κB activation upon stimulation with TNF (FIG. 5) or IL-1considerably. This suggests that ABIN mediates the NF-κB inhibitingeffect of A20.

Total ABIN (1-647; SEQ ID NO:2), the shorter splice variant (54-647; SEQID NO:19) and the C-terminal fragment (390-647) are also able to blockthe TPA induced NF-κB activation (FIG. 6).

Similar results were obtained when ABIN2 was used in the test instead ofABIN or ABIN-fragments (FIG. 7).

Example 6

Effect of ABIN and/or ABIN Fragments on NF-κB Activation Induced byOverexpression of TRADD, RIP, TRAF2, NIK or p65.

Expression vectors for ABIN and ABIN fragments were constructed asdescribed above. The expression vectors containing TRAF2, NIK and p65have previously been described (Malinin et al., 1997; Rothe et al.,1994; Vanden Berghe et al., 1998). PCR fragments encoding TRADD and RIPwere cloned in pCDNA3 (Invitrogen, Carlsbad, Calif.) in frame with aC-terminal E-tag. Transfection and reporter assay was carried out asdescribed above.

NF-κB can be activated in 293 T cells by TNF treatment as well as byoverexpression of specific proteins of the TNF-receptor complex,including TRADD, RIP, TRAF2 and NIK (Rothe et al.,1995; Malinin et al.,1997; Hsu et al., 1995; Ting et al., 1996). The latter associates withand activates IκB kinase complex which leads to IκB phosphorylation.This is a signal for ubiquitination and degradation of IκB, thusreleasing NF-κB which then translocates to the nucleus. Co-transfectionof expression plasmids encoding these TNF-receptor associated proteinstogether with the expression plasmids encoding full length ABIN, showedthat the latter completely inhibited NF-κB activation induced by TRADDor RIP, and partially inhibited TRAF2-induced NF-κB activation. Incontrast, no clear difference was observed when NF-κB-dependent reportergene expression was induced by NIK or more directly by overexpression ofthe p65 subunit of NF-κB (FIG. 8, FIG. 9). These results suggest thatABIN inhibits TNF-induced NF-κB activation at a level preceding theactivation of the NIK-IκB kinase steps, for example at the level ofTRAF2 in the TNF-receptor complex.

As members of the TRAF family mediate NF-κB activation by several otherstimuli, including IL-1, lymphotoxin β, CD30 and CD40 (Rothe et al.,1995; Cao et al., 1996; Nakano et al., 1996; Aizawa et al., 1997; Ishadaet al., 1996), ABIN might have the potential to inhibit NF-κB activationin response to a wide range of inducers. Therefore, drugs that mimic theactivity of ABIN are likely to have therapeutic value in inflammatoryand neurodegenerative diseases as well as in cancer and AIDS.

Example 7 Cell Transfection, Co-immunoprecipitation and WesternBlotting.

2×10⁶ human embryonic kidney 293T cells were plated on 10 cm Petridishes and transiently transfected by calcium phosphate-DNAcoprecipitation. 24 hours after transfection, cells were lysed in 500 lof lysis buffer (50 mM Hepes pH 7.6, 250 mM NaCl, 0.1% Nonidet P-40 and5 mM EDTA). Lysates were incubated with 5 l of rabbit anti-GFP antibody(Clontech) and immunocomplexes were immobilized on protein A-Trisacryl(Pierce). The latter were washed twice with lysis buffer and twice withlysis buffer containing 1M NaCl. Coprecipitating proteins were separatedby SDS-PAGE and analyzed by Western blotting with mouse anti-E-tagantibody (Pharmacia).

Example 8 NF-κB Dependent Reporter Gene Assay.

NF-κB activity was determined by the NF-κB dependent expression of aluciferase reporter gene. Therefore, 293T cells were plated in 6-wellplates at 4×10⁵ cells per well and transiently transfected by thecalcium phosphate-DNA coprecipitation method. Each transfectioncontained 800 ng of the specific expression plasmids, as well as 100 ngof pNFconluc plasmid and 100 ng pUT651 plasmid. 24 hours aftertransfection, these transfectants were trypsinized and seeded on a24-well plate. Another 24 hours later, cells were either stimulated with1000 IU/ml hTNF or 7000 IU/ml IL-1 or left untreated. After 6 hours ofstimulation, cells were lysed in 200 μl lysis buffer and analyzed forluciferase and β-galactosidase activity. Luciferase values (luc) arenormalized with (β-galactosidase values (gal) and plotted as luc/gal.

Example 9 Site Specific Mutagenesis.

Site specific mutagenesis on ABIN was performed by overlap PCR reactionusing primers which contain the desired mutations. The primers used werethe mutation primers5′-GAATACCAGGAGGCGCAGATCCAGCGGCTCAATAAAGCTTTGGAGGAGGC-3′ (SEQ ID NO:11),5′-GTTGCTGAAAGAGGACGTCAAAATCTTTGAAGAGG-3′ (SEQ ID NO:12),5′-GCAGGTAAAAATCTTTGAAGAGAATGCCCAGAGGGAACG-3′ (SEQ ID NO:13), and5′-GCAGGTAAAAATCTTTGAAGAGGACTTCCAGAGGGAAC GGAGTGATGCGCAACGCATGCCCG-3′(SEQ ID NO:14), a forward primer located at the start codon and tworeverse primers, one hybridizing in the 3′ UTR and one in the codingregion. The XhoI-BstEII fragment of wild type ABIN(54-647) in pCAGGS wasexchanged with the same fragment of the PCR amplified mutated ABINcDNA's.

Example 10 Binding of ABIN With A20 is Not Sufficient for its NF-κBInhibiting Potential.

A two hybrid assay with A20 revealed another novel A20-binding proteinwhich was also able to inhibit NF-κB activation upon overexpression.BLAST searches with this novel protein, named ABIN-2, revealed nohomology with any known protein. However, by comparison of the proteinsequence of ABIN-2 with ABIN, two boxes of 19 (AA 423-441) and 21 (AA475-495) amino acids long with 68% and 67% homology were identifiedrespectively. Therefore, the contribution of these regions to thebinding with A20 and to the NF-κB inhibiting effects of ABIN wereanalyzed by site specific mutagenesis of a number of conserved aminoacids. Co-immunoprecipitation analysis after transient overexpression ofGFP or GFP/A20 together with wild type ABIN or its site specific mutants(ABIN-MUT1, ABIN-MUT2, ABIN-MUT3 and ABIN-MUT4) (SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17 and SEQ ID NO:18 respectively) in 293T cells, showedthat all of these mutants can still bind A20 (FIG. 10). On the otherhand, point mutations in the second box (ABIN-MUT2, ABIN-MUT3 andABIN-MUT4) completely abolished the ability of ABIN to block NF-κBactivation upon stimulation of 293T cells with TNF, even when higheramounts of these expression plasmids were transfected. In contrast, themutation in the first box (ABIN-MUT1) only slightly diminished the NF-κBinhibiting effect of ABIN (FIG. 11). Furthermore, point mutations in thesecond conserved motif dominantly interfered with the NF-κB inhibitingeffect of wild type ABIN (FIG. 12). ABIN-MUT2 and ABIN-MUT3 exhibit amore potent function as dominant negative mutants of ABIN, compared toABIN-MUT4. In these assays, comparable expression levels of thedifferent mutants and of wild type ABIN were obtained as judged byWestern blot analysis using anti-E tag antibody. These results suggestthat the second conserved region is involved in the NF-κB inhibitingeffects of ABIN, and that binding of ABIN with A20 as such is notsufficient for inhibition of NF-κB activation.

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1. A method of screening a compound for its ability to activate orsuppress ABIN (A20-Binding Inhibitor of NF-κB activation) dependentNF-κB inhibition, said method comprising: a) combining a compound to bescreened with a protein comprising ABIN amino acid consensus sequence ofSEQ ID NO: 9 and having the ability to interact with protein A20, b)detecting an interaction between said compound and said protein, c)identifying compounds that interact with said protein, d) obtaining acell line with that nucleic acid sequence encoding protein A20, nucleicacid sequence encoding said ABIN consensus sequence protein, and anNF-κB dependent reporter gene, e) administering at least one of TNF(tumor necrosis factor), IL-1 (interleukin-1), TPA (tissue plasminogenactivator), TRADD (TNF receptor associated death domain), RIP (receptorinteracting protein), TRAF2 (TNF receptor associated factor 2) to thecell line to induce activation of the NF-κB pathway, f) administeringthe detected compounds to said cell line, and g) determining if theadministration of the detected compounds alter NF-κB dependent reportergene expression, wherein an increase in expression indicates that thedetected compounds suppress ABIN dependent NF-κB inhibition and adecrease in expression indicates that the compounds activate ABINdependent NF-κB inhibition.
 2. The method according to claim 1, whereindetecting an interaction between said compound and said proteincomprises using either a two-hybrid assay or a co-immunoprecipitationassay.
 3. A method of screening a compound for its ability to activateor suppress ABIN (A20-Binding Inhibitor of NF-κB activation) dependentNF-κB inhibition, said method comprising: a) combining a compound to bescreened with a protein comprising ABIN amino acid consensus sequence ofSEQ ID NO:9 and having the ability to inhibit NF-κB activation, b)detecting an interaction between said compound and said protein, c)identifying compounds that interact with said protein, d) obtaining acell line with a nucleic acid sequence encoding said ABIN consensussequence protein and an NF-κB dependent reporter gene, e) administeringto the cell line an inducer of the NF-κB pathway, wherein the inductionof the NF-κB pathway is inhibitable by said ABIN consensus sequenceprotein, f) administering one of the detected compounds to said cellline, and g) determining if the administration of the detected compoundalters NF-κB dependent reporter gene expression, wherein an increase inexpression indicates that the detected compound suppresses ABINdependent NF-κB inhibition and a decrease in expression indicates thatthe detected compound activates ABiN dependent NF-κB inhibition.
 4. Themethod according to claim 3, wherein obtaining a cell line comprisesobtaining a cell line including a nucleic acid sequence encoding proteinA20.
 5. The method according to claim 3, wherein the inducer of theNF-κB pathway comprises at least one of TNF (tumor necrosis factor),IL-1 (interleukin-1), TPA (tissue plasminogen activator), TRADD (TNFreceptor associated death domain), RIP (receptor interacting protein),or TRAF2 (TNF receptor associated factor 2).
 6. A method of screening acompound for its ability to activate or suppress ABIN (A20-BindingInhibitor of NF-κB activation) dependent NF-κB inhibition, said methodcomprising: a) combining a compound to be screened with a proteincomprising ABIN amino acid consensus sequence of SEQ ID NO:9 and havingthe ability to inhibit NF-κB activation, b) detecting an interactionbetween said compound and said protein, c) identifying compounds thatinteract with said protein, d) obtaining a cell line with a nucleic acidsequence encoding said ABIN consensus sequence protein, an NF-κBdependent reporter gene, and a NF-κB pathway inducible by at least oneof TNF (tumor necrosis factor), IL-1 (interleukin-1), TPA (tissueplasminogen activator), TRADD (TNF receptor associated death domain),RIP (receptor interacting protein), or TRAF2 (TNF receptor associatedfactor 2), e) inducing activation of the NF-κB pathway of said cellline, f) administering one of the detected compounds to said cell line,and g) determining if the administration of the detected compound altersNF-κB dependent reporter gene expression, wherein an increase inexpression indicates that the detected compound suppresses ABINdependent NF-κB inhibition and a decrease in expression indicates thatthe detected compound activates ABIN dependent NF-κB inhibition.